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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
Route of exposure unphysiological - results of limited use for risk assessment and classification

Data source

Reference
Reference Type:
publication
Title:
Prenatal Exposure to Carbon Black (Printex 90): Effects on Sexual Development and Neurofunction
Author:
Jackson P., Vogel U., Wallin H., Hougard KS
Year:
2011
Bibliographic source:
Nanotoxicology, August 2012; 6(5): 486–500

Materials and methods

Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
The study assessed whether maternal pulmonary exposure to Printex 90 affects sexual development and neurofunction in the prenatally exposed offspring. Time-mated mice were intratracheally instilled with Printex 90 dispersed in Millipore water on gestation days (GD) 7, 10, 15 and 18, with total doses of 11, 54 and 268 lg Printex 90⁄ animal.
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
- Declared Particle size: 14 nm (Degussa Hüls)- Geometric mean size: 65 nm- Morphology: Individual carbon black spheres mainly occurred in open-structured long chain aggregates and fewer large dense aggregates- Surface area: 295 -338 m2/g- Pycnometric particle density: 2.1 g/cm3- Chemical composition 99% C, 0.8% N and 0.01% H2- The total PAH content (Carbon black extract – Soxhlet): 0.0742 µg/g- The total PAH content (DEP extract – NIST SRM 1650): 216 µg

Test animals

Species:
mouse
Strain:
other: C57BL/ 6BomTac
Details on test animals and environmental conditions:
TEST ANIMALS - Source: Taconic Europe, Ejby, DK - Age at study initiation: not specified - Weight at study initiation: yes; see table 4- Received as time-mated pregnant mice on gestation day 3 - Acclimation period: tests were commenced on gestation day 8:

Administration / exposure

Route of administration:
intratracheal
Vehicle:
water
Remarks:
g-irradiated Nanopure Diamond UV water (Pyrogens: < 0.001 EU/ml, Total Organic Carbon: < 3.0 ppb),
Details on exposure:
The particle preparation and instillation procedures were described previously. Printex 90 was sonicated for 8 min (10 s pulses and 10 s pauses, total sonication time 4 min) at a concentration of 1.675 mg/mL (67 mg/instillation) in 0.2 mm filtered, g-irradiated Nanopure Diamond UV water (Pyrogens: < 0.001 EU/ml, Total Organic Carbon: < 3.0 ppb), using a 400 W Branson Sonifier S-450D (Branson Ultrasonics Corp., Danbury, CT, USA) mounted with a disruptor horn and operated at 10% amplitude. This dispersion was used for the high dose and diluted 1:5 for the medium dose (13.4 mg/ instillation) and diluted futher 1:5 for the low dose (2.7 mg/instillation). Eighty time-mated mice were anesthetized with 3% Isoflurane and instilled with a vehicle or one of the three concentrations of Printex 90 dispersions (40 mL solution followed by 160 mL air) on GD 7, 10, 15 and 18. We chose to instill Printex 90 at times that would cover the major part of the fetal development. We tried to distribute the dose over that period assuming that a fraction of the particles would have been cleared rapidly, but that much of the dose would remain in the lungs for several weeks. Exposure took place between 08:30 and 14:30 h. Time-mated mice were instilled in different order each day, to reduce any variation that might be related to the time of exposure. The total instilled doses were 11, 54 and 268 mg/animal.
Analytical verification of doses or concentrations:
no
Remarks:
not applicable
Details on analytical verification of doses or concentrations:
The particle size distribution in the Printex 90 dispersions was determined with a 633 nm He- Ne Dynamic Laser Scatter (DLS) Zetasizer nano ZS (Malvern Inc., UK).The dispersion of Printex 90 instillation fluid was also analyzed by Scanning Electron Microscopy (QUANTA 200 FEG MKII with EDX).The particle size distribution was similar in the three instilled dispersions with concentrations of 1675, 335 and 67 mg/mL (see Figure 3), and was stable for more than 1 h. The average zeta-size was approximately 140 nm and the hydrodynamic number size-distributions had a peak size between 50 and 60 nm When converted to volume distributions, minor amounts of mm-size particles and two smaller size modes with peak sizes around 50–60 nm and 200–400 nm were identified. The observed DLS sizes were confirmed by TEM and SEM, with a wide size distribution of nm- to mm-size free and agglomerated particles. The agglomerates consisted of spherical to sub-spherical carbonaceous particles as well as minor amounts of free single primary spheres.
Details on mating procedure:
no data. Time-mated nulliparous adult female mice were employed.
Duration of treatment / exposure:
Instillations on day 7, 10, 15, 18
Frequency of treatment:
single instillation per day
Duration of test:
no data
Doses / concentrationsopen allclose all
Dose / conc.:
11 other: µg/animal
Remarks:
2.7 µg/instillation administered over 4 instillations
Dose / conc.:
54 other: µg/animal
Remarks:
13.4 µg/instillation administered over 4 instillations
Dose / conc.:
268 other: µg/animal
Remarks:
67 µg/instillation administered over 4 instillations
No. of animals per sex per dose:
Control: 2411& 54 µg/animal: 17268 µg/animal: 22
Control animals:
yes, concurrent vehicle
Details on study design:
After the last exposure on GD 18, the time-mated mice were housed alone and monitored for birth. The expected day of delivery, GD 20, was assigned as post-natal day zero (PND 0) for the offspring. At weaning, offspring were randomly distributed into balanced experimental groups with one female and one male per litter (where possible): a group for collection of organs on PND 23 (in Jackson et al 2012b), a group for sexual development data (all dose groups) and a group for behavioural testing (control and 268 µg Printex 90 ⁄ animal). Females and males were housed separately in cages of four or five (extra animals were added when needed).

Examinations

Maternal examinations:
Maternal inflammation was assessed by monitoring neutrophil influx in the lungs.
Fetal examinations:
Anogenital distance (AGD) was measured with a slide gauge in all offspring at weaning. Relative AGD was calculated (AGD⁄ cube root of body-weight). Results are reported as litter average separately for female and male offspring. The onset of puberty (vaginal opening in females and pre-putial separation in males) was recorded three times a week, between PND 26 and 40. The offspring were weighed on PND 32, 39 and 47.Behavioural testing was performed on PND 72–75, during the light period with experimenters blinded to exposure status. The same observer was used within tests, and exposed and control animals were tested alternately. Animals were transferred to the experimental room 1 hr before the first test. Activity was assessed for 3 min in a circular (B = 1 m) Open field. Total ambulation and ambulation in 1-min time bins (to test for habituation) were calculated. Duration in each of the Open field zones (central and peripheral) and the number of zone crossings were extracted. Acoustic startle reaction (ASR) and pre-pulse inhibition (PPI) were tested. The offspring group assigned to behavioural testing was weighed on PND 39, 53, 67 and 81.
Statistics:
The accepted level of statistical significance was 0.05. Litter was considered the statistical unit, where relevant. Data were analysed by analyses of variance (ANOVA), with treatment and sex as factors. When relevant, analysis of repeated measures was applied (days of sampling in PND, trials and time bins). Significant results from overall analyses were analysed by pairwise comparisons. Analyses were performed in SYSTAT 9. Puberty start was analysed by log rank testin SAS 9.2.
Historical control data:
no data

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
not specified
Details on results:
Persistent lung inflammation was induced in the exposed time-mated mice (the number of neutrophil cells in bronchoalveolar lavage fluid was increased 28-fold 3 days after exposure and 61-fold 26 days after exposure

Maternal developmental toxicity

Details on maternal toxic effects:
Details on developmental maternal toxicity are described in Jackson et al 2012b. Briefly, no effects on gestation, lactation were observed.

Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
NOAEC
Effect level:
268 other: µg/animal
Based on:
test mat.
Basis for effect level:
other: Gestational and post gestational parameters
Key result
Dose descriptor:
LOAEC
Effect level:
268 other: µg/animal
Based on:
test mat.
Basis for effect level:
other: Persistent lung inflammation

Maternal abnormalities

Key result
Abnormalities:
effects observed, treatment-related
Localisation:
other: respiratory system
Description (incidence and severity):
persistent increase of inflammatory markers in BAL

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): Weight gain after weaning was similar in exposed offspring and their controls, both in the groups observed for sexual maturation and in the group designated for behavioural testing
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
not specified
Changes in postnatal survival:
no effects observed
External malformations:
not examined
Skeletal malformations:
not examined
Visceral malformations:
not examined
Description (incidence and severity):
Relative AGD measured on all offspring at weaning did not differ between offspring prenatally exposed to Printex 90 and their controls. Overall analysis of onset of puberty (vaginal opening) in females indicated statistically significant differences between groups. Female offspring prenatally exposed to 11 lg Printex 90⁄ animal entered puberty significantly earlier (time of vaginal opening, p = 0.01) compared with controls, while higher-dose groups compared with thecontrols. As data did not indicate dose response this may be a chance finding. Puberty start in the male offspring (time of pre-putial separation) started at similar time in the exposed groups and in the controls.The basal acoustic startle response was of similar magnitude in exposed and control offspring, and the offspring reacted similarly to PPI. For locomotor activity investigated in the Open field, the overall statistical analysis indicated no difference in the total distance moved during the 3-min observation. However, analysis of offspring habituation in minute intervals as repeated measure in the ANOVA indicated that the pattern differed between groups. The females moved differently during the first 2 min, compared with their control; total movement during the first minute was reduced compared with that of control (p = 0.03); and total movement during the second minute was increased compared with that of control (p = 0.03). Thus, the female offspring prenatally exposed to 268 µg Printex 90⁄ animal displayed a different pattern of habituation. There were no differences in Open field activity between the exposed and control male offspring

Effect levels (fetuses)

open allclose all
Key result
Dose descriptor:
NOEC
Effect level:
54 other: µg/animal
Based on:
test mat.
Sex:
female
Basis for effect level:
other: changed habituation pattern
Key result
Dose descriptor:
NOEC
Effect level:
268 other: µg/animal
Based on:
test mat.
Sex:
male
Basis for effect level:
other: neurobehavioural change

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
268 other: µg/animal
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
not specified

Applicant's summary and conclusion

Conclusions:
Despite using very high intratracheal doses, gestational and litter parameters were normal for dams and offspring in a study evaluating the effects of carbon black on the sexual development and neurofunction of mice exposed in-utero to carbon black up to a total dose of 54 µg//animal. The female offspring prenatally exposed to 268 µg/animal displayed altered habituation pattern during the Open Field test.
Executive summary:

Time-mated mice were intratracheally instilled with Printex 90 dispersed in Millipore water on gestation days 7, 10, 15 and 18, with total doses of 11, 54 and 268 µg Printex 90/animal. The female offspring prenatally exposed tgo 268 µg/animal displayed altered habituation pattern during the Open Field test.